The differential expression of the cellular src-gene product pp60src and its phosphokinase activity in normal chicken cells and tissues

Chick embryos of different ages and adult chickens were examined for the expression of pp60c -src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. In any brain extract, the pp60c -src kinase activity was always high, whereas muscle extracts of embryos show an age-dependent decrease in kinase activity. Adult animals show either no or barely measurable activity in muscle tissue. In contrast, liver cell extracts of embryos show an age-dependent increase in pp60c -src kinase activity, with adult chickens displaying the highest activity, very similar to that found in brain extracts. This demonstrates that increased expression of c-src is not necessarily correlated with cell proliferation, but suggests that, at an early stage of differentiation of mesenchymal cells, the relatively high expression of c-src could be responsible, at least in part, for the control of cell metabolism and proliferation.     

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells.

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.     

Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src

The retroviral oncogene v-src arose by transduction of the cellular gene c-src. The similarity between these genes raised the possibility that c-src might be able to elicit neoplastic growth. We explored this by constructing a chimeric plasmid that allows the expression of chicken c-src. A rat cell line containing ten times the normal intracellular level of pp60c -src was isolated after transfecting rat-2 cells with the chimeric DNA. These cells produce the protein encoded by c-src ( pp60c -src) in quantities at least three times greater than required to achieve transformation by the product of v-src ( pp60v -src). The cells remain phenotypically normal, contain actin cables, and do not grow in soft agar. However, transfection of the cell line containing elevated cells of pp60c -src or Rat-2 cells with a molecular clone of v-src produces cells that exhibit properties of biologically transformed cells: round morphology, disrupted actin cables, and ability to grow in soft agar.     

Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin

The derivation and characterization of 22 hybridoma clones producing monoclonal antibodies (Mabs) specific for the transforming protein of Rous sarcoma virus, pp60src, are described. All Mabs reacted with pp60v-src encoded by Prague, Schmidt-Ruppin, and Bratislava 77 strains of Rous sarcoma virus. Of these Mabs, 10 efficiently immunoprecipitated pp60c-src from chicken embryo cells. Of these 10 Mabs, 2 (GD11 and EB8) readily detected pp60c-src from a variety of rodent and human cultured cells and from rat brain tissue in an in vitro immune complex kinase assay. Mapping experiments have tentatively localized the determinant(s) recognized by GD11 and EB8 to a region of the src protein bounded by amino acid residues 82 to 169, whereas the remaining Mabs appeared to recognize determinants residing within residues 1 to 82 or 169 to 173. Most of the Mabs complexed denatured pp60v-src in a Western immunoblot, and several were used to localize pp60v-src in Rous sarcoma virus-transformed chicken embryo cells by indirect immunofluorescence microscopy.     

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.     

Rous sarcoma virus variants that encode src proteins with an altered carboxy terminus are defective for cellular transformation.

The src gene of Rous sarcoma virus (v-src) and its cellular homolog, the c-src gene, share extensive sequence homology. The most notable differences between these genes reside in the region encoding the carboxy terminus of the src proteins. We constructed mutations within the 3' end of the v-src gene to determine the significance of this region to the transforming potential of the v-src protein, pp60v-src. The mutants CHdl300 and CHis1511 contain mutations that alter the last 23 amino acids of pp60v-src, whereas the mutant CHis1545-C contains a linker insertion that alters the last 11 amino acids of pp60v-src, and the mutant CHis1545-H contains a linker insertion that results in a 9-amino-acid insertion at position 415. Plasmids bearing each of these mutations were unable to transform chicken cells when introduced into these cells by DNA transfection. In addition, the structurally altered src proteins encoded by the mutants had much-reduced levels of tyrosine protein kinase activity in vivo, as measured by autophosphorylation and phosphorylation of the 34,000-Mr cellular protein, and in vitro, as determined by measuring the level of pp60src autophosphorylation. These data indicate that the carboxy-terminal amino acid sequences play an important role in maintaining the structure of the catalytic domain of pp60v-src. In contrast, the transfection of chicken cells with plasmid DNA containing a chimeric v-c-src gene resulted in morphological cell transformation and the synthesis of an enzymatically active hybrid protein. Therefore, the carboxy-terminal sequence alterations observed in the c-src protein do not alone serve to alter the functional activity of a hybrid v-c-src protein appreciably.     

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation

We examine the interaction between polyoma-virus-encoded middle tumor antigen and the cellular src gene product, pp60c-src, using a series of monoclonal antibodies that recognize mammalian pp60c-src. Our results show that infection of mouse cells with transformation-competent strains of polyoma virus results in the stimulation of pp60c-src kinase activity severalfold over that observed in uninfected mouse cells and mouse cells infected with transformation-deficient polyoma virus. A similar degree of enhancement of pp60c-src kinase activity was found in polyoma-virus-transformed rodent cells. No differences were detected in the level of pp60c-src synthesis in polyoma-virus-infected and uninfected mouse cells or polyoma-virus-transformed and normal rodent cells. These studies demonstrate that polyoma-virus-encoded middle tumor antigen is associated with pp60c-src in lysates of polyoma-virus-infected and polyoma-virus-transformed cells and suggest a novel mechanism for the functional activation of a cellular proto-oncogene product, namely, that the interaction between middle tumor antigen and pp60c-src leads to a stimulation of pp60c-src tyrosyl kinase activity.     

pp60c-src is developmentally regulated in the neural retina

We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.     

Development and characterization of antisera specific for amino- and carboxy-terminal regions of pp60src.

Antisera to the Rous sarcoma virus transforming protein pp60src were produced in rabbits immunized with p60src purified from recombinant Escherichia coli. Immunization with isolated V8 proteolytic fragments of p60src resulted in antisera specific for the amino- and carboxy-terminal domains. The reactions of these antisera with pp60v-src and the normal cell protein p60c-src were characterized.     

5''-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.     

Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts.

The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.     

Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases.

A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp60v-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gag-fps) and Yamaguchi 73 avian sarcoma virus (P90gag-yes), both of which are tyrosine-specific protein kinases. However, we found that the endogenous c-src product, pp60c-src, was not tightly bound to the detergent-insoluble matrix. This does not appear to have been due to differences in the cytoskeleton between transformed and nontransformed cells since pp60c-src was also solubilized by nonionic detergent in cells transformed by Rous sarcoma virus. This difference in the affinities of the v-src and c-src products for cytoskeletal proteins may contribute to the inability of pp60c-src to transform cells.     

Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo.

We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.     

Activation of the pp60c-src kinase during differentiation of monomyelocytic cells in vitro.

The proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60c-src tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60c-src kinase.